This invention concerns methods and devices for separating nucleic acid from substances to which it is bound non-specifically utilizing combined direct and alternating field electrophoresis. The devices and methods are useful for rapid diagnosis of disease.
The discovery of the helical structure of DNA made possible the comparison of DNA in the genomes of various species. It was soon discovered that in the cells of each species there are segments of DNA comprised of nucleotide sequences unique to that species. Furthermore it was found that viruses also contain unique segments of DNA. These unique segments provide the basis for a new technology and promise a new solution to the problem of detecting living organisms in biological specimen. They will be especially important in the diagnosis of diseases caused by pathogenic organisms (Falkow, S. et al. U.S. Pat. No. 4,348,535).
In this technology a "probe", a segment of single-stranded nucleic acid which is complementary to a specific DNA, is used to identify a cell or virus in a sample. The probe is put into contact with the sample and is allowed to react by base pairing to form a double-stranded hybrid with any complementary single-stranded DNA which is present. This hybrid is then separated from the sample and any unreacted probe. Reaction is followed by means of a label, a radioactive or chromophoric group, for example, on the hybrid.
Separation of hybrid from probe has been a fundamental obstacle in the development of this technology. This step, as presently practiced, is time consuming, laborious, and may lead to erroneous results.
Initial efforts to separate hybrid and probe have been directed towards immobilizing the hybrid on an inert surface which can be washed to remove probe from the sample being analyzed. It was known that DNA in solution, in its naturally occurring double-stranded form, can be denatured, that is, separated into two separate strands, and then again renatured simply by manipulating the temperature as well as the ionic strength and pH of the solution. Single-stranded DNA can similarly be caused to react with single-stranded RNA in solution to form a double-stranded hybrid. (C. L. Schildkraut, J. Marmur, J. Fresco and P. Doty, J. Biol. Chem. 236, 803 (1961; B. D. Hall and S. Spiegelman, Proc. Nat'l. Acad. Sci. USA 47, 137 (1961). Gillespie and Spiegelman (D. Gillespie and S. Spiegelman, J. Mol. Biol. 12, 829 (1965)) were able to immobilize single-stranded DNA on nitrocellulose and found that the immobilized DNA was able to react with DNA or RNA in solution to form an immobilized hybrid. This became the basis of a workable separation technique. Other researchers, notably Bernardi (G. Bernardi, Nature 206, 779 (1965)) and Kohne (D. E. Kohne, Biophys. J. 8, 1104 (1968)) found that hydroxyl apatite 15 binds double-stranded hybrids but not nucleic acid. They used the hydroxyl apatite to "fish" hybrids from solution.
These two traditional methods of separating double-stranded nucleic acid hybrids from single-stranded probes have aided the study of nucleic acids in the laboratory, but have not proven useful in clinics where repeated diagnoses must be performed. Long times are required for each analysis which involves multiple washing steps and other manipulations, and a high level of skill is needed to make the methods operative.
Because of the inherent problems of the immobilization technique, a novel enzymatic approach was attempted. It was found that S1 nuclease is capable of digesting single-stranded chains of RNA and DNA into nucleotides, but the enzyme does not attack the double-stranded forms (Leong, J. A., Gerapin, A. C., Jackson, N., Fanshier, Ll, Levinson, W. and Biship, J. M. (1972) J. Virol., 891). The enzyme may thus be used to remove interfering single-stranded probes from the hybrid. This procedure has proven useful in the hands of researchers "with good hands" but has not found acceptance in more routine applications because the enzyme is difficult to control, is sensitive to small buffer changes and results have been difficult to reproduce.
Rosbash (Rosbash, M., et al., In "Methods in Enzymology" Vol. 68, pp. 454-469 (1979)) has attempted to separate nucleic acids by utilizing their size differences. He was able to separate single-stranded from double-stranded nucleic acids by gel filtration on agarose beads. However, this method, again, is mostly a research technique since artifactual complexes such as background human DNA interfere with separation. Also, the size difference between single- and double-strands must be large to allow efficient separation. Although temperature jacketing increases separation, with this modification the method becomes too clumsy for routine use.
Schwarz and Cantor have used electrophoretic methods to separate large segments of DNA. They found, while analyzing chromosomes, that large segments of DNA may be separated from one another on an agarose bed by applying discontinuous pulsed field electric gradients to the agarose bed. This method is useful for separating large nucleic acid strands, but has not been useful for separating probes from hybrids.
In a hybridization assay, the small probes form true hybrids with a specific segment of nucleic acid through the binding of complementary base pairs, but the probes also tend to bind indiscriminately by other non-specific bonds to the larger setgments and also to other larger components normally found in biological specimen. There is no way in conventional electrophoretic methods, as there is in methods involving immobilized hybrids, to wash away the artifactual probe. As a result, false bands appear on the gel.
Accordingly, a simple, effective, rapid method for hybridization assays with effectual separation of artifactual nucleic acid probe from larger aggregates and hybrids has been sought.